Small- and intermediate-conductance Ca -activated K channels directly control agonist-evoked nitric oxide synthesis in human vascular endothelial cells

Sheng J-Z, Braun AP. Smalland intermediate-conductance Ca -activated K channels directly control agonist-evoked nitric oxide synthesis in human vascular endothelial cells. Am J Physiol Cell Physiol 293: C458–C467, 2007. First published April 25, 2007; doi:10.1152/ajpcell.00036.2007.—The contribution of small-conductance (SKCa) and intermediate-conductance Ca -activated K (IKCa) channels to the generation of nitric oxide (NO) by Ca mobilizing stimuli was investigated in human umbilical vein endothelial cells (HUVECs) by combining single-cell microfluorimetry with perforated patch-clamp recordings to monitor agonist-evoked NO synthesis, cytosolic Ca transients, and membrane hyperpolarization in real time. ATP or histamine evoked reproducible elevations in NO synthesis and cytosolic Ca , as judged by 4-amino-5-methylamino-2 ,7 -difluorofluorescein (DAF-FM) and fluo-3 fluorescence, respectively, that were tightly associated with membrane hyperpolarizations. Whereas evoked NO synthesis was unaffected by either tetraethylammonium (10 mmol/l) or BaCl2 (50 mol/l) ouabain (100 mol/l), depleting intracellular Ca stores by thapsigargin or removing external Ca inhibited NO production, as did exposure to high (80 mmol/l) external KCl. Importantly, apamin and charybdotoxin (ChTx)/ triarylmethane (TRAM)-34, selective blockers SKCa and IKCa channels, respectively, abolished both stimulated NO synthesis and membrane hyperpolarization and decreased evoked Ca transients. Apamin and TRAM-34 also inhibited an agonist-induced outwardly rectifying current characteristic of SKCa and IKCa channels. Under voltage-clamp control, we further observed that the magnitude of agonist-induced NO production varied directly with the degree of membrane hyperpolarization. Mechanistically, our data indicate that SKCa and IKCa channel-mediated hyperpolarization represents a critical early event in agonist-evoked NO production by regulating the influx of Ca responsible for endothelial NO synthase activation. Moreover, it appears that the primary role of agonist-induced release of intracellular Ca stores is to trigger the opening of both KCa channels along with Ca entry channels at the plasma membrane. Finally, the observed inhibition of stimulated NO synthesis by apamin and ChTx/TRAM-34 demonstrates that SKCa and IKCa channels are essential for NO-mediated vasorelaxation.